A wide variety of tests is used for the detection and for the quantitation of biomolecules. A high sensitivity is required for many of these as they are present in biological fluids in very small concentration. Furthermore, the assays must have a high degree of selectivity in order to be able to determine a specific biomolecule in the presence of other entities.
Many tests are based on the coupling of the species to be determined to another moiety and the subsequent determination of the labeled entity. There exist various assays based on the interaction of enzymes and their substrates; on the interaction of antigens and antibodies, between hormones and receptors, etc. There exists a wide variety of assays which are based on radioactive labels and on other types of labels.
The translation of selective interaction into a measurable quantity often implies coupling of a detectable labeling to one or more of the interacting species. Radioisotope labeling is one kind of such labeling which is used in analytical clinical laboratory.
However, the desire to avoid the use of radioactive techniques has stimulated the development of other labels. Among these enzymes appear to be practical: an enzyme is coupled with the bioactive material as a marker and the enzyme activity is measured. Enzyme labels increase the sensitivity through chemical amplification. Chemical amplification refers to the passing of a substance through a catalytic cycling, or multiplication mechanism to generate a relatively large amount of product. The rate at which the product is formed is related to the concentration of the analyte in the sample. The enzyme activity is usually measured by optical instruments such as colorimeters or spectrophotometers. The development of electrochemical bioassays has receive only little attention. Electrochemical methods are free of sample turbidity quenching, and interferences from the many absorbing and fluorescing compounds in typical biological samples that hinder spectroscopic techniques. Enzyme electrodes are an example of the combination of enzyme action with electrochemical measurements for analytical purposes. Enzyme electrodes are a type of biosensors. Biosensors are analytical devices in which biological materials capable of specific chemical recognition, are in intimate contact with transducers. Among these, bioelectrochemical sensors such as enzyme electrodes have found promising application especially in clinical and process measurements. Commercial analyzers equipped with enzyme sensors are available mostly for serum components measurements. There have been several attempts to construct other biosensors such as e.g. immunosensors.
However, at present amongst the disadvantages of such sensors are the following: The sensitivity is low as compared with established methods such as enzyme immuno-assays and a measuring cell is occupied for the whole incubation time to form an antigen-antibody complex. Therefore the advantages of electrochemical sensors-their short response time-is not exploited efficiently. Only a few samples can be measured per day and fast measurements are impossible. This is true especially in clinical tests which usually involve a large number of samples. The use of a single electrode and the calibration it requires prior to measurements impedes the measurements and does not allow its successful application in clinical tests in hospitals and clinics.
The system can be used to carry out assays with a wide variety of biointeractions. The invention is illustrated in the following with reference to a number of specific examples, which are to be construed in a non-limitative manner.
One of the representative enzymes which provide for a wide scope of substrates is alkaline phosphatase.
The enzyme alkaline phosphatase enzyme is a common label in immunological tests. Conjugates of antigens and antibodies with this enzyme are commercially available in a rather purified form. The common substrates for alkaline phosphatase used in various immunological tests are nitrophenyl phosphate and phenylphosphate. Electrochemical determinations of alkaline phosphatase based on the hydrolysis products of the enzyme reaction are rather difficult because of the high over-voltage of the phenol compounds and serious problems arising from adsorption to the electrode and fouling of the electrode response.